Peptide inhibitors of leukocyte adhesion

ABSTRACT

The present invention provides novel peptides derived from portions of the sequence of amino acids 42-48 of P-selectin. The invention also provides pharmaceutical compositions comprising the peptides of the invention, and diagnostic and therapeutic methods utilizing the peptides and pharmaceutical compositions of the invention.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 07/941,652,filed Sep. 8, 1992 now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to peptides which inhibit binding of selectinssuch as P-selectin, E-selectin and L-selectin.

The adherence of platelets and leukocytes to vascular surfaces is acritical component of the inflammatory response and is part of a complexseries of reactions involving the simultaneous and interrelatedactivation of the complement, coagulation, and immune systems.

The complement proteins collectively play a leading role in the immunesystem, both in the identification and in the removal of foreignsubstances and immune complexes, as reviewed by Muller-Eberhard, H. J.,Ann. Rev. Biochem. 57: 321-347 (1988). Central to the complement systemare the C3 and C4 proteins, which when activated covalently attach tonearby targets, marking them for clearance. In order to help controlthis process, a remarkable family of soluble and membrane-boundregulatory proteins has evolved, each of which interacts with activatedC3 and/or C4 derivatives. The coagulation and inflammatory pathways areregulated in a coordinate fashion in response to tissue damage. Forexample, in addition to becoming adhesive for leukocytes, activatedendothelial cells express tissue factor on the cell surface and decreasetheir surface expression of thrombomodulin, leading to a netfacilitation of coagulation reactions on the cell surface. In somecases, a single receptor can be involved in both inflammatory andcoagulation processes.

Leukocyte adherence to vascular endothelium is a key initial step inmigration of leukocytes to tissues in response to microbial invasion.Although a class of inducible leukocyte receptors, the CD11-CD18molecules, are thought to have some role in adherence to endothelium,mechanisms of equal or even greater importance for leukocyte adherenceappear to be due to inducible changes in the endothelium itself.

Activated platelets have also been shown to interact with bothneutrophils and monocytes in vitro. The interaction of platelets withmonocytes may be mediated in part by the binding of thrombospondin toplatelets and monocytes, although other mechanisms have not beenexcluded. The mechanisms for the binding of neutrophils to activatedplatelets are not well understood, except that it is known that divalentcations are required. In response to vascular injury, platelets areknown to adhere to subendothelial surfaces, become activated, andsupport coagulation. Platelets and other cells may also play animportant role in the recruitment of leukocytes into the wound in orderto contain microbial invasion.

Endothelium exposed to "rapid" activators such as thrombin and histaminebecomes adhesive for neutrophils within two to ten minutes, whileendothelium exposed to cytokines such as tumor necrosis factor andinterleukin-1 becomes adhesive after one to six hours. The rapidendothelial-dependent leukocyte adhesion has been associated withexpression of the lipid mediator platelet activating factor (PAF) on thecell surface, and presumably, the appearance of other endothelialsurface receptors. The slower cytokine-inducible endothelial adhesionfor leukocytes is mediated, at least in part, by E-selectin that issynthesized by endothelial cells after exposure to cytokines and thentransported to the cell surface, where it binds neutrophils. Theisolation, characterization and cloning of E-selectin or ELAM-1 isreviewed by Bevilacqua, et al., in Science 243, 1160-1165 (1989).L-selectin, a peripheral lymph node homing receptor, also called "themurine Mel 14 antigen", "Leu 8", the "Leu 8 antigen" and "LAM-1", isanother structure on neutrophils, monocytes, and lymphocytes that bindslymphocytes to high endothelial venules in peripheral lymph nodes. Thecharacterization and cloning of the protein is reviewed by Lasky, etal., Cell 56, 1045-1055 (1989) (mouse) and Tedder, et al., J. Exp. Med.170, 123-133 (1989).

P-selectin, also known as GMP-140 (granule membrane protein 140), orPADGEM, is a cysteine-rich and heavily glycosylated integral membraneglycoprotein with an apparent molecular weight of 140,000 as assessed bysodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).P-selectin was first purified from human platelets by McEver and Martin,J. Biol. Chem. 259: 9799-9804 (1984). The protein is present in alphagranules of resting platelets but is rapidly redistributed to the plasmamembrane following platelet activation, as reported by Stenberg, et al.,J. Cell Bio. 101, 880-886 (1985). The presence of P-selectin inendothelial cells and its biosynthesis by these cells was reported byMcEver, et al., Blood 70(5) Suppl. 1:355a, Abstract No. 1274 (1987). Inendothelial cells, P-selectin is found in storage granules known as theWeibel-Palade bodies. (McEver, et al. J. Clin. Invest. 84: 92-99 (1989)and Hattori, et al., J. Biol. Chem. 264: 7768-7771 (1989)). P-selectin(called GMP-140 or PADGEM) has also been reported to mediate theinteraction of activated platelets with neutrophils and monocytes byLarsen, et al., in Cell 59, 305-312 (October 1989) and Hamburger andMcEver, Blood 75: 550-554 (1990).

The cDNA-derived amino acid sequence, reported by Johnston, et al., inCell 56, 1033-1044 (Mar. 24, 1989), and in U.S. Ser. No. 07/320,408filed Mar. 8, 1989, indicates that it contains a number of modulardomains that are likely to fold independently. Beginning at theN-terminus, these include a "lectin" domain, an "EGF" domain, ninetandem consensus repeats similar to those in complement bindingproteins, a transmembrane domain (except in a soluble form that appearsto result from differential splicing), and a cytoplasmic tail.

When platelets or endothelial cells are activated by mediators such asthrombin, the membranes of the storage granules fuse with the plasmamembrane, the soluble contents of the granules are released to theexternal environment, and membrane bound P-selectin is presented withinseconds on the cell surface. The rapid redistribution of P-selectin tothe surface of platelets and endothelial cells as a result of activationsuggested that this glycoprotein could play an important role at sitesof inflammation or vascular disruption.

This important role has been confirmed by the observation thatP-selectin is a receptor for neutrophils (Geng et al., Nature343:757-760 (1990); Hamburger and McEver, Blood 75:550-554 (1990)),monocytes (Larsen, et al. Cell 59:305-312 (1989)); Moore, et al., J.Cell Biol. 112:491-499 (1991)), and perhaps a subset of lymphocytes(Moore, et al. J. Cell Biol. 112:491-499 (1991)). Thus, GMP-140 canserve as a receptor for leukocytes following its rapid mobilization tothe surfaces of platelets and endothelial cells stimulated with agonistssuch as thrombin. This role in leukocyte recruitment may be important inhemostatic and inflammatory processes in both physiologic and pathologicstates.

Peptides derived from P-selectin are described in U.S. Ser. No.07/554,199 entitled "Functionally Active Selectin-Derived Peptides"filed Jul. 17, 1990 by Rodger P. McEver that are useful in diagnosticsand in modulating the hemostatic and inflammatory responses in a patientwherein a therapeutically effective amount of a peptide capable ofblocking leukocyte recognition of P-selectin is administered to thepatient. U.S. Ser. No. 07/554,199 filed Jul. 17, 1990 also disclosesthat peptide sequences within the lectin domain of P-selectin, havinghomology with the lectin domains of other proteins, especiallyE-selectin and the L-selectin, selectively inhibit neutrophil adhesionto purified P-selectin, and can therefore be used in diagnostic assaysof patients and diseases characterized by altered binding by thesemolecules, in screening assays for compounds altering this binding, andin clinical applications to inhibit or modulate interactions ofleukocytes with platelets or endothelial cells involving coagulationand/or inflammatory processes.

E-selectin, L-selectin, and P-selectin have been termed "selecting",based on their related structure and function. E-selectin is not presentin unstimulated endothelium. However, when endothelium is exposed tocytokines such as tumor necrosis factor of interleukin-1, the gene forE-selectin is transcribed, producing RNA which in turn is translatedinto protein. The result is that E-selectin is expressed on the surfaceof endothelial cells one to four hours after exposure to cytokines, asreported by Bevilacqua et al., Proc.Natl.Acad.Sci.USA 84: 9238-9242(1987) (in contrast to P-selectin, which is stored in granules andpresented on the cell surface within seconds after activation).E-selectin has been shown to mediate the adherence of neutrophils tocytokine-treated endothelium and thus appears to be important inallowing leukocytes to migrate across cytokine-stimulated endotheliuminto tissues. The cDNA-derived primary structure of E-selectin indicatesthat it contains a "lectin" domain, an EGF domain, and six (instead ofthe nine in P-selectin) repeats similar to those ofcomplement-regulatory proteins, a transmembrane domain, and a shortcytoplasmic tail. There is extensive sequence homology betweenP-selectin and E-selectin throughout both proteins, but the similarityis particularly striking in the lectin and EGF domains.

Homing receptors are lymphocyte surface structures that allowlymphocytes to bind to specialized endothelial cells in lymphatictissues, termed high endothelial cells or high endothelial venules(reviewed by Yednock and Rose, Advances in Immunology, vol. 44, F. I.Dixon, ed., 313-378 (Academic Press, New York 1989). This binding allowslymphocytes to migrate across the endothelium into the lymphatic tissueswhere they are exposed to processed antigens. The lymphocytes thenre-enter the blood through the lymphatic system. L-selectin, alymphocyte homing receptor, contains a lectin domain, an EGF domain, twocomplement-binding repeats, a transmembrane domain, and a shortcytoplasmic tail. L-selectin also shares extensive sequence homologywith P-selectin, particularly in the lectin and EGF domains.

Based on a comparison of the lectin domains between P-selectin,E-selectin, and L-selectin, it may be possible to select those peptidesinhibiting binding of neutrophils to P-selectin which will inhibitbinding of E-selectin, L-selectin, and other homologous selecting, tocomponents of the inflammatory process, or, conversely, which willinhibit only P-selectin binding.

The in vivo significance of platelet-leukocyte interactions has not beenstudied carefully. However, in response to vascular injury, plateletsare known to adhere to subendothelial surfaces, become activated, andsupport coagulation. Platelets and other cells may also play animportant role in the recruitment of leukocytes into the wound in orderto contain microbial invasion. Conversely, leukocytes may recruitplatelets into tissues at sites of inflammation, as reported byIssekutz, et al., Lab. Invest. 49:716 (1983).

The coagulation and inflammatory pathways are regulated in a coordinatefashion in response to tissue damage. For example, in addition tobecoming adhesive for leukocytes, activated endothelial cells expresstissue factor on the cell surface and decrease their surface expressionof thrombomodulin, leading to a net facilitation of coagulationreactions on the cell surface. In some cases, a single receptor can beinvolved in both inflammatory and coagulation processes.

Proteins involved in the hemostatic and inflammatory pathways are ofinterest for diagnostic purposes and treatment of human disorders.However, there are many problems using proteins therapeutically.Proteins are usually expensive to produce in quantities sufficient foradministration to a patient. Moreover, there can be a reaction againstthe protein after it has been administered more than once to thepatient. It is therefore desirable to develop peptides having the same,or better, activity as the protein, which are inexpensive to synthesize,reproducible and relatively innocuous.

It is preferable to develop peptides which can be preparedsynthetically, having activity at least equal to, or greater than, thepeptides derived from the protein itself.

It is therefore an object of the present invention to provide peptidesinteracting with cells recognized by selecting, including P-selectin,E-selectin, and L-selectin.

It is another object of the present invention to provide methods forusing these peptides to inhibit leukocyte adhesion to endothelium or toplatelets.

It is a further object of the present invention to provide methods forusing these peptides to modulate the immune response and the hemostaticpathway.

It is yet another object of the present invention to provide peptidesfor use in diagnostic assays relating to P-selectin, E-selectin andL-selectin.

SUMMARY OF THE INVENTION

It has now been found that peptides of the selectin 36-50 sequence areeffective in inhibiting the adhesion of leukocytes, especiallyneutrophils, to selecting.

This invention relates to novel peptides having a formula selected fromFormulas I, II and III:

    R.sup.1 --X.sup.1 --A'--B'--C'--D'--E'--F'--G'--Y.sup.1 --R.sup.2 (I)

    R.sup.1 --X.sup.2 --cyclo(Z.sup.1 --Z.sup.2 --Z.sup.3 --Z.sup.4 --A'--B'--C'--D'--E'--F'--Z.sup.5)--Y.sup.2 --R.sup.2     (II)

    R.sup.2 --X.sup.3 --cyclo(Z.sup.1 --B'--C'--D'--E'--Z.sup.5)--Y.sup.3 --R.sup.2                                                 (III)

or a pharmaceutically acceptable acid- or base-addition salt thereof,wherein:

A' is D- or L-leucine, D- or threoine, or D- or L-glutamine;

B' is D- or L-proline, D- or L-serine, D- or L-alanine, D- or L-valine,D- or L-isoleucine, D- or L-leucine, 2-aminobutyric acid, or null(signifying no amino acid);

C' is D- or L-tyrosine, D- or L-proline, D- or L-arginine, or D- orL-alanine;

D' is D- or L-tyrosine, D- or L-phenylalanine, D- or L-serine orglycine;

E' is D- or L-serine, D- or L-threonine, or D- or L-proline;

F' is D- or L-serine, D- or L-tyrosine, D- or L-alanine, or D- orL-threonine;

G' is D- or L-tyrosine, or D- or L-alanine;

X¹, X² and X³ are each, independently, a linear chain of from 0 to 6amino acids;

Y¹, Y² and Y³ are each, independently, a linear chain of from 0 to 3amino acids;

Z¹ and Z⁵ are independently selected from the group consisting of D- orL-cysteine, D- or L-homocysteine, D- or L-aspartic acid, D- orL-glutamic acid, D- or L-lysine, D- or L-3-amino propionic acid, D- orL-4-amino butyric acid, and D- or L-5-amino valeric acid, provided that(a) when one of Z¹ and Z⁵ is a cysteine or homo-cysteine, the other isalso independently a cysteine or homo-cysteine; (b) when one of Z¹ andZ⁵ is an aminodicarboxylic acid, then the other is a diaminodicarboxylicacid;

Z² is D- or L-asparagine;

Z³ is D- or L-lysine;

Z⁴ is D- or L-valine, D- or L-proline, D- or L-serine, D- or L-asparticacid, or glycine;

R¹ is H (signifying a free-terminal amino group), formyl, lower alkyl,aryl, lower alkanoyl, aroyl, alkyloxycarbonyl, aryloxycarbonyl ordesamino (signifying no alpha amino group on the N-terminal amino acid);and

R² is H (signifying descarboxy where the α carboxyl group in theC-terminal amino acid is absent), OR³, or NR⁴ R⁵ ;

R³ is H (signifying a free carboxylic acid on a C-terminal carboxylgroup) or lower alkyl or aryl; and

R⁴ and R⁵ are each selected independently from H, lower alkyl, and aryl,or taken together are a methylene chain of 4-8 methylene groups(--(CH₂)_(n) -- where n=4 to 8);

provided that, when X'--A'--B'--C'--D'--E'--F'--G'--Y' isDYLNKVLPYYSSYYW, then R² is NH₂.

Peptides of the invention have as their core region portions of the42-48 amino acid sequences of the selectins, with residue 1 defined asthe N-terminus of the mature proteins after the cleavage of the signalpeptides.

The peptides of Formulas II and III are cyclic by virtue of theformation of a disulfide bond between cysteine residues or an amide bondbetween side chain groups of diaminocarboxylic acid and anaminodicarboxylic acid. The general techniques for the formation of thisbond are described by G. Barany and R. B. Merrifield in The PeptidesAnalysis, Synthesis, Biology, (Academic Press, Inc., 1979), as well asin other reference works known to those skilled in the art.

Tests indicate that peptides of the invention inhibit the binding ofneutrophils to P-selectin in concentrations of peptide ranging fromabout 1 to about 1500 μm. Tests also indicate that alterations withinthe core sequence, including the addition or deletion of amino acids, donot result in loss of biological activity.

This invention relates not only to the novel peptides of Formulas I, IIand III, but also to pharmaceutical compositions comprising them, todiagnostic and therapeutic methods utilizing them, and to methods ofpreparing them.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ability of the peptides of Example 1,Glu-Tyr-Leu-Asn-Ser-Ile-Leu-Ser-Tyr-Ser-Pro-Ser-Tyr-Tyr-Trp-NH₂ (SEQ IDNO:23), and the peptide of Example II,Asp-Tyr-Leu-Asn-Lys-Val-Leu-Pro-Tyr-Tyr-Ser-Ser-Tyr-Tyr-Val-NH₂ (SEQ IDNO:1), to inhibit the binding of human neutrophils to human P-selectinin a dose-dependent manner.

DETAILED DESCRIPTION OF THE INVENTION

Preferred peptides of this invention are those of Formula I aspreviously defined, wherein X¹ is selected from a group consisting of:

Glu-Tyr-Leu-Asn-Ser-Ile,

Asp-Tyr-Leu-Asn-Lys-X⁴, where X⁴ is selected from the group consistingof Val, Pro, Gly, Ser and Asp;

Tyr-Leu-Asn-Lys-Val,

Leu-Asn-Lys-Val,

Asn-Lys-Val,

Asp-Val,

Lys-Val,

Asn-Thr,

Val, Lys or null (signifying no amino acid),

wherein each of the above-designated amino acids may be a D- or L- aminoacid.

Additional preferred peptides of this invention are those of Formula IIwhere X² is selected from the group consisting of Asp-Tyr, Tyr, and null(signifying no amino acid), wherein each of the amino acids may be a D-or L-amino acid.

Still addition preferred peptides of the invention are those of FormulaIII where X³ is selected from the group consisting of

Asp-Tyr-Leu-Asn-Lys-Val

Tyr-Leu-Asn-Lys-Val

Leu-Asn-Lys-Val

Asn-Lys-Val, and

Lys-Val,

wherein each of the amino acids may be a D- or L- amino acid.

Also preferred are peptides having Formula I as previously defined,wherein Y¹ is selected from a group consisting of Tyr, Tyr-Val, Tyr-Trp,X⁵ -X⁶ -Val-, where X⁵ and X⁶ are each independently an aromatic orhydrophobic amino acid; and null (signifying no amino acid), where eachof the amino acids may be a D- or L- amino acid.

Additional preferred peptides are those of Formula II where Y² isselected from the group consisting of Tyr-Val, Tyr, and null (signifyingno amino acid), wherein each of the amino acids may be a D- or L- aminoacid.

Still additional preferred peptides are those of Formula III where Y³ isselected from the group consisting of Tyr-Tyr-Val, Tyr-Tyr, Tyr, andnull (signifying no amino acid), wherein each of the amino acids may bea D- or L-amino acid.

Representative examples of specifically preferred peptides include thefollowing "Preferred Peptides" (SEQ ID NOS:1-42):

    __________________________________________________________________________    (SEQ ID NO: 1)            Asp--Tyr--Leu--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--T            yr--Val-NH.sub.2 ;    (SEQ ID NO: 2)            Tyr--Leu--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr--V            al-NH.sub.2 ;    (SEQ ID NO: 3)            Leu--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr--Val-NH            .sub.2 ;    (SEQ ID NO: 4)            Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr--Val-NH.sub.            2 ;    (SEQ ID NO: 5)            Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr--Val-NH.sub.2 ;    (SEQ ID NO: 6)            Asp--Tyr--Leu--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--T            yr-NH.sub.2 ;    (SEQ ID NO: 7)            Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 8)            Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 9)            Lys--Thr--Leu--Pro--Phe--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 10)            Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr--Val-NH.sub.2 ;    (SEQ ID NO: 11)            Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr-NH.sub.2 ;    (SEQ ID NO: 12)            Lys--Val--Leu--Ala--Tyr--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 13)            Lys--Val--Leu--Pro--Ala--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 14)            Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ala--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 15)            Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Ala--Tyr-NH.sub.2 ;    (SEQ ID NO: 16)            Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Thr--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 17)            Lys--Val--Leu--Pro--Tyr--Tyr--Thr--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 18)            Asp--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 19)            Lys--Val--Leu--Pro--Tyr--Gly--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 20)            Lys--Val--Leu--Pro--Arg--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 21)            Asn--Thr--Leu--Pro--Tyr--Ser--Pro--Tyr--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 22)            Lys--Val--Gln--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 23)            Glu--Tyr--Leu--Asn--Ser--Ile--Leu--Ser--Tyr--Ser--Pro--Ser--Tyr--T            yr--Trp-NH.sub.2 ;    (SEQ ID NO: 24)            Asp--Tyr-cyclo(Cys--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--C            ys)--Tyr--Val-NH.sub.2 ;    (SEQ ID NO: 25)            Tyr-cyclo(Cys--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Cys)--            Tyr--Val-NH.sub.2 ;    (SEQ ID NO: 26)            Asp--Tyr-cyclo(Cys--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--C            ys)--Tyr-NH.sub.2 ;    (SEQ ID NO: 27)            Asp--Tyr-cyclo(Cys--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--C            ys)-NH.sub.2 ;    (SEQ ID NO: 28)            Tyr-cyclo(Cys--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Cys)--            Tyr-NH.sub.2 ;    (SEQ ID NO: 29)            cyclo(Cys--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Cys)--Tyr-            -Val-NH.sub.2 ;    (SEQ ID NO: 30)            cyclo(Cys--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Cys)--Tyr-            NH.sub.2 ;    (SEQ ID NO: 31)            cyclo(Cys--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Cys)-NH.su            b.2 ;    (SEQ ID NO: 32)            Asp--Tyr--Leu--Asn--Lys--Val-cyclo(Cys--Pro--Tyr--Tyr--Ser--Cys)--            Tyr--Tyr--Val-NH.sub.2 ;    (SEQ ID NO: 33)            Tyr--Leu--Asn--Lys--Val-cyclo(Cys--Pro--Tyr--Tyr--Ser--Cys)--Tyr--            Tyr--Val-NH.sub.2 ;    (SEQ ID NO: 34)            Leu--Asn--Lys--Val-cyclo(Cys--Pro--Tyr--Tyr--Ser--Cys)--Tyr--Tyr--            Val-NH.sub.2 ;    (SEQ ID NO: 35)            Asn--Lys--Val-cyclo(Cys--Pro--Tyr--Tyr--Ser--Cys)--Tyr--Tyr--Val-N            H.sub.2 ;    (SEQ ID NO: 36)            Lys--Val-cyclo(Cys--Pro--Tyr--Tyr--Ser--Cys)--Tyr--Tyr--Val-NH.sub            .2 ;    (SEQ ID NO: 37)            Asp--Tyr--Leu--Asn--Lys--Val-cyclo(Cys--Pro--Tyr--Tyr--Ser--Cys)--            Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 38)            Asp--Tyr--Leu--Asn--Lys--Val-cyclo(Cys--Pro--Tyr--Tyr--Ser--Cys)--            Tyr-NH.sub.2 ;    (SEQ ID NO: 39)            Asp--Tyr--Leu--Asn--Lys--Val-cyclo(Cys--Pro--Tyr--Tyr--Ser--Cys)-N            H.sub.2 ;    (SEQ ID NO: 40)            Tyr--Leu--Asn--Lys--Val-cyclo(Cys--Pro--Tyr--Tyr--Ser--Cys)--Tyr--            Tyr-NH.sub.2 ;    (SEQ ID NO: 41)            Leu--Asn--Lys--Val-cyclo(Cys--Pro--Tyr--Tyr--Ser--Cys)--Tyr--Tyr-N            H.sub.2 ;    (SEQ ID NO: 42)            Leu--Asn--Lys--Val-cyclo(Cys--Pro--Tyr--Tyr--Ser--Cys)--Tyr-NH.sub            .2.    __________________________________________________________________________

Table I shows the ability of certain preferred peptides to inhibitbinding of human neutrophils to human P-selectin at a peptideconcentration of 300 μM.

                  TABLE I    ______________________________________                            % INHIBITION                            AT 0.3 μM    STRUCTURE               PEPTIDE CONC.    ______________________________________    DYLNKVLPYYSSYYV-NH.sub.2                    (SEQ ID NO: 1)                                71    YLNKVLPYYSSYYV-NH.sub.2                    (SEQ ID NO: 2)                                101    LNKVLPYYSSYYV-NH.sub.2                    (SEQ ID NO: 3)                                100    NKVLPYYSSYYV-NH.sub.2                    (SEQ ID NO: 4)                                80    KVLPYYSSYYV-NH.sub.2                    (SEQ ID NO: 5)                                102    DYLNKVLPYYSSYY-NH.sub.2                    (SEQ ID NO: 6)                                61    VLPYYSSYY-NH.sub.2                    (SEQ ID NO: 7)                                11    LPYYSSYY-NH.sub.2                    (SEQ ID NO: 8)                                15    KTLPFSSYY-NH.sub.2                    (SEQ ID NO: 9)                                33    VLPYYSSYYV-NH.sub.2                    (SEQ ID NO: 10)                                66    VLPYYSSY-NH.sub.2                    (SEQ ID NO: 11)                                25    KVLAYYSSYY-NH.sub.2                    (SEQ ID NO: 12)                                68    KVLPAYSSYY-NH.sub.2                    (SEQ ID NO: 13)                                20    KVLPYYSAYY-NH.sub.2                    (SEQ ID NO: 14)                                18    KVLPYYSSAY-NH.sub.2                    (SEQ ID NO: 15)                                12    KVLPYYSTYY-NH.sub.2                    (SEQ ID NO: 16)                                22    KVLPYYTSYY-NH.sub.2                    (SEQ ID NO: 17)                                32    DVLPYYSSYY-NH.sub.2                    (SEQ ID NO: 18)                                57    KVLPYGSSYY-NH.sub.2                    (SEQ ID NO: 19)                                34    KVLPRYSSYY-NH.sub.2                    (SEQ ID NO: 20)                                19    NTLPYSPYYY-NH.sub.2                    (SEQ ID NO: 21)                                39    KVQPYYSSY Y-NH.sub.2                    (SEQ ID NO: 22)                                29    EYLNSILSYSPSYYW-NH.sub.2                    (SEQ ID NO: 23)                                54    ______________________________________

As used herein, the term "alkyl" includes branched, straight-chain, andcyclic saturated hydrocarbons. The term "lower alkyl" means an alkylhaving from one to six carbon atoms, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,cyclopentylmethyl and hexyl. The term "alkanoyl" means ##STR1## whereinR⁶ is a alkyl group.

The term "aroyl" means ##STR2##

wherein R⁷ is an aryl group. The term "aryl" means an aromatic orheteroaromatic structure having between one and three rings, which mayor may not be ring fused structures, and are optionally substituted withhalogens, carbons, or other heteroatoms such as nitrogen (N), sulfur(S), phosphorus (P), and boron (B).

The term alkoxycarbonyl means ##STR3##

wherein R⁸ is a lower alkyl group.

The term aryloxycarbonyl means ##STR4##

wherein R⁹ is an aryl and arylmethyl group.

Halogen refers to fluorine, chlorine, bromine or iodine.

The term "terminal α-amino group of X" refers to the α-amino group ofthe N-terminal amino acid of X.

The peptides of the invention can be used in the form of the freepeptide or a pharmaceutically acceptable salt. Amine salts can beprepared by treating the peptide with an acid according to knownmethods. Suitable acids include inorganic acids such as hydrochloricacid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, and phosphoric acid, and organic acids such as formicacid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvicacid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaricacid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, andsulfanilic acid.

Carboxylic acid groups in the peptide can be converted to a salt bytreating the peptide with a base according to known methods. Suitablebases include inorganic bases such as sodium hydroxide, ammoniumhydroxide, and potassium hydroxide, and organic bases such as mono-,di-, and tri-alkyl and aryl amines (e.g., triethylamine,diisopropylamine, methylamine, and dimethylamine and optionallysubstituted mono-, di, and tri-ethanolamines.

As referred to herein, the amino acid components of the peptides andcertain materials used in their preparation are identified byabbreviations for convenience. These abbreviations are as follows:

    ______________________________________    Amino Acid    Abbreviations    ______________________________________    L-alanine          Ala         A    D-alanine          D-Ala       a    L-arginine         Arg         R    D-arginine         D-Arg       r    D-asparagine       D-Asn       N    L-asparagine       Asn         n    L-aspartic acid    Asp         D    D-aspartic acid    D-Asp       d    L-cysteine         Cys         C    D-cysteine         D-Cys       c    L-glutamic acid    Glu         E    D-glutamic acid    D-Glu       e    L-glutamine        Gln         Q    D-glutamine        D-Gln       q    glycine            Gly         G    L-histidine        His         H    D-histidine        D-His       h    L-isolelucine      Ile         I    D-isolelucine      D-Ile       i    L-leucine          Leu         L    D-leucine          D-Leu       l    L-lysine           Lys         K    D-lysine           D-Lys       k    L-phenylalanine    Phe         F    D-phenylalanine    D-Phe       f    L-proline          Pro         P    D-proline          D-Pro       p    L-pyroglutamic acid                       pGlu    D-pyroglutamic acid                       DpGlu    L-serine           Ser         S    D-serine           D-Ser       s    L-threonine        Thr         T    D-threonine        D-Thr       t    L-tyrosine         Tyr         Y    D-tyrosine         D-Tyr       y    L-tryptophan       Trp         W    D-tryptophan       D-Trp       w    L-valine           Val         V    D-valine           D-Val       v    L-alloisolucine    Allo    D-alloisolucine    D-Allo    ______________________________________    Reagents           Abbreviations    ______________________________________    Trifluoroacetic acid                       TFA    Methylene chloride CH.sub.2 Cl.sub.2    N,N-Diisopropylethylamine                       DIEA    N-Methylpyrrolidone                       NMP    1-Hydroxybenzotriazole                       HOBT    Dimethylsulfoxide  DMSO    Acetic anhydride   Ac.sub.2 O    Diisopropylcarbodiimide                       DIC    ______________________________________

Amino acids preceded by L- or D- refer, respectively, to the L- or D-enantiomer of the amino acid, whereas amino acids not preceded by L- orD- refer to the L- enantiomer.

The term "aromatic amino acid" as used herein means an amino acidcontaining, or substituted with, an aryl group. Aromatic amino acidsinclude phenylalanine, tyrosine, tryptophan, histidine, andnaphthylalanine.

The term "hyrophobic amino acid" as used herein means amino acidscontaining non-polar groups that tend to decrease water solubility.Hydrophobic amino acids include, but are not limited to, leucine,isoleucine, valine, phenylalanine, alanine and naphthylalanine.

Methods of Preparation of Peptides

The peptides can generally be prepared following known techniques, asdescribed, for example, in the cited publications, the teachings ofwhich are specifically incorporated herein. In a preferred method, thepeptides are prepared following the solid-phase synthetic techniqueinitially described by Merrifield in J.Amer.Chem.Soc., 85, 2149-2154(1963). Other techniques may be found, for example, in M. Bodanszky, etal, Peptide Synthesis, second edition, (John Wiley & Sons, 1976), aswell as in other reference works known to those skilled in the art.

Appropriate protective groups usable in such syntheses and theirabbreviations will be found in the above text, as well as in J. F. W.McOmie, Protective Groups in Organic Chemistry, (Plenum Press, New York,1973). The common protective groups used herein are t-butyloxycarbonyl(Boc), fluorenylmethoxycarboyl (FMOC), benzyl (Bzl), tosyl (Tos),o-bromo-phenylmethoxycarbonyl (BrCBZ), phenylmethoxycarbonyl (CBZ),2-chloro-phenylmethoxycarbonyl (2-Cl-CBZ),4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), trityl (Trt), formyl(CHO), and tertiary butyl (t-Bu).

General synthetic procedures for the synthesis of peptides of theinvention by solid phase methodology are as follows:

    ______________________________________                     REPETITIONS                              TIME    ______________________________________    A. General Synthetic Procedures For Solid Phase Peptide    Synthesis Using N.sup.α -Boc Protection    1.  25% TFA in CH.sub.2 Cl.sub.2                           1          3 min.    2.  50% TFA in CH.sub.2 Cl.sub.2                           1          16 min.    3.  CH.sub.2 Cl.sub.2  5          3 min.    4.  5% DIEA in NMP     2          4 min.    5.  NMP                6          5 min.    6.  Coupling step      1          57 min.        a. Preformed BOC-Amino Acid-  37 min.         HOBT active ester in NMP        b. DMSO                       16 min.        c. DIEA                       5 min.    7.  10% Ac.sub.2 O, 5% DIEA in NMP                           1          9 min.    8.  CH.sub.2 Cl.sub.2  5          3 min.    B. General Synthetic Procedure For Solid Phase Peptide    Synthesis Using N.sup.α -FMOC Protection    1.  50% piperidine in NMP                           1          1 min.    2.  50% piperidine in NMP                           1          12 min.    3.  NMP wash           7          1 min.    4.  Coupling           2          30 min.    FMOC amino acid dissolved in HOBT/NMP followed by    the addition of DIC (di-isopropylcarbodiamide) in    NMP.    5.  NMP wash.          2          1 min.    ______________________________________

N-terminal acetylation on the deprotected N.sup.α -amino group ofpeptides synthesized using either Boc or FMOC strategies is accomplishedwith 10% Ac₂ O and 5% DIEA in NMP, followed by washing of the peptideresin with NMP and/or CH₂ Cl₂.

The peptides can also be prepared using standard genetic engineeringtechniques known to those skilled in the art. For example, the peptidecan be produced enzymatically by inserting nucleic acid encoding thepeptide into an expression vector, expressing the DNA, and translatingthe DNA into the peptide in the presence of the required amino acids.The peptide is then purified using chromatographic or electrophoretictechniques, or by means of a carrier protein which can be fused to, andsubsequently cleaved from, the peptide by inserting into the expressionvector in phase with the peptide encoding sequence a nucleic acidsequence encoding the carrier protein. The fusion protein-peptide may beisolated using chromatographic, electrophoretic or immunologicaltechniques (such as binding to a resin via an antibody to the carrierprotein). The peptide can be cleaved using chemical methodology orenzymatically, as by, for example, hydrolases.

Peptides of the invention can also be prepared using solution methods,by either stepwise or fragment condensations. An appropriately alphaamino-protected amino acid is coupled to an appropriately alpha carboxylprotected amino acid (such protection may not be required depending onthe coupling method chosen) using diimides, symmetrical or unsymmetricalanhydrides, BOP, or other coupling reagents or techniques known to thoseskilled in the art. These techniques may be either or enzymatic. Thealpha amino and/or alpha carboxyl protecting groups are removed and thenext suitably protected amino acid or block of amino acids are coupledto extend the growing peptide. Various combinations of protecting groupsand of chemical and/or enzymatic techniques and assembly strategies canbe used in each synthesis.

Methods of Preparation of Pharmaceutical Compositions

Pharmaceutical compositions of this invention comprise apharmaceutically acceptable carrier or diluent and an effective quantityof one or more of the peptides of the invention or an acid or base saltthereof. The carrier or diluent may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.,sublingual, rectal, nasal, oral, or parenteral.

In preparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed, for example, waters, oils,alcohols, flavoring agents, preservatives, and coloring agents, to makean oral liquid preparation (e.g., suspension, elixir, or solution) orwith carriers such as starches, sugars, diluents, granulating agents,lubricants, binders, and disintegrating agents, to make an oral solidpreparation (e.g., powder, capsule, or tablet).

Controlled release forms or enhancers to increase bioavailability mayalso be used. Because of their ease in administration, tablets andcapsules represent the most advantageous oral dosage unit form, in whichcase solid pharmaceutical carriers are employed. If desired, tablets maybe sugar coated or enteric coated by standard techniques.

For parenteral products, the carrier will usually be sterile water,although other ingredients to aid solubility or as preservatives may beincluded. Injectable suspensions may also be prepared, in which caseappropriate liquid carriers and suspending agents can be employed.

The peptides can also be administered locally at a wound or inflammatorysite by topical application of a solution or cream.

Alternatively, the peptide may be administered in liposomes ormicrospheres (or microparticles). Methods for preparing liposomes andmicrospheres for administration to a patient are known to those skilledin the art. U.S. Pat. No. 4,789,734 describes methods for encapsulatingbiological materials in liposomes. Essentially, the material isdissolved in an aqueous solution, the appropriate phospholipids andlipids added, along with surfactants if required, and the materialdialyzed or sonicated, as necessary. A review of known methods is by G.Gregoriadis, Chapter 14, "Liposomes", Drug Carriers in Biology andMedicine, pp. 287-341 (Academic Press, 1979). Microspheres formed ofpolymers or proteins are well known to those skilled in the art, and canbe tailored for passage through the gastrointestinal tract directly intothe bloodstream. Alternatively, the peptide can be incorporated and themicrospheres, or composite of microspheres, implanted for slow releaseover a period of time, ranging from days to months. See, for example,U.S. Pat. Nos. 4,906,474, 4,925,673 and 3,625,214.

The peptides are generally active when administered parenterally inamounts above about 1 μg/kg body weight. The peptides are generallyactive when administered parenterally in amounts above about 1 μg/kgbody weight. Effective doses by other routes of administration aregenerally those which result in similar blood level to i.v. doses aboveabout 1 μg/Kg. For treatment to prevent organ injury in cases involvingreperfusion, the peptides may be administered parenterally in amountsfrom about 0.01 to about 10 mg/kg body weight. Generally, the same rangeof dosage amounts may be used in treatment of other diseases or ofconditions where inflammation is to be reduced. This dosage will bedependent, in part, on whether one or more peptides are administered.

Methods for Demonstrating Binding

Peptides that are biologically active are those which inhibit binding ofneutrophils, monocytes, subsets of lymphocytes or other cells toP-selectin, or which inhibit leukocyte adhesion to endothelium that ismediated by ELAM-1 and/or the homing receptor.

Peptides can be screened for their ability to inhibit adhesion to cells,for example, neutrophil adhesion to purified P-selectin immobilized onplastic wells, using the assay described by Geng, et al., Nature 343,757-760 (1990).

Human neutrophils are isolated from heparinized whole blood by densitygradient centrifugation on Mono-Poly resolving media, Flow Laboratories.Neutrophil suspensions are greater than 98% pure and greater than 95%viable by trypan blue exclusion. For adhesion assays, neutrophils aresuspended at a concentration of 2×10⁶ cells/mL in Hanks' balanced saltsolution containing 1.26 mM Ca²⁺ and 0.81 mM Mg²⁺ (HBSS, Gibco) with gmg/mL human serum albumin (HBSS/HSA). Adhesion assays are conducted intriplicate in 96-well microtiter plates, Corning, incubated at 4° C.overnight with 50 microliters of various protein solutions.

P-selectin is isolated from human platelet lysates by immunoaffinitychromatography on antibody S12-Sepharose™ and ion-exchangechromatography on a Mono-Q™ column (FLPC, Pharmacia Fine Chemicals), asfollows.

Outdated human platelet packs (100 units) obtained from a blood bank andstored at 4° C. are pooled, adjusted to 5 mM EDTA at pH 7.5, centrifugedat 4,000 rpm for 30 minutes in 1 liter bottles, then washed three timeswith 1 liter of 0.1 M NaCl, 20 mM Tris pH 7.5 (TBS), 5 mM EDTA, 5 mMbenzamidine.

The pellets are then resuspended in a minimum amount of wash buffer andmade 1 mM in DIFP, then frozen in 50 mL screwtop tubes at -80° C. Thefrozen platelets are thawed and resuspended in 50 mL TBS, 5 mMbenzamidine, 5 mM EDTA pH 7.5, 100 M leupeptin. The suspension is frozenand thawed two times in a dry ice-acetone bath using a 600 mLlyophilizing flask, then homogenized in a glass/teflon mortar and pestleand made 1 mM in DIFP. The NaCl concentration is adjusted to 0.5 M witha stock solution of 4 M NaCl. After stirring the suspension at 4° C., itis centrifuged in polycarbonate tubes at 33,000 rpm for 60 minutes at 4°C. The supernatant (0.5 M NaCl wash) is removed and saved; thissupernatant contains the soluble form of P-selectin. Care is taken notto remove the top part of the pellet with the supernatant. The pelletsare then homogenized in extraction buffer (TBS, 5 mM benzamidine, 5 mMEDTA, pH 7.5, 100 μM leupeptin, 2% Triton X-100). After centrifugationat 19,500 rpm for 25 minutes at 4° C., the supernatant is removed. Theextraction procedure is repeated with the pellet and the supernatant iscombined with the first supernatant. The combined extracts, whichcontain the membrane form of P-selectin, are adjusted to 0.5 M NaCl.

The soluble fraction (0.5 M NaCl wash) and the membrane extract (alsoadjusted to 0.5 M NaCl) are absorbed with separate pools of themonoclonal antibody S12 (directed to P-selectin) previously coupled toAffigel (Biorad) at 5 mg/mL for 2 hours at 4° C. After letting theresins settle, the supernatants are removed. The S12 Affigel containingbound GMP-140 is then loaded into a column and washed overnight at 4° C.with 400 mL of 0.5 M NaCl, 20 mM Tris pH 7.5, 0.01% Lubrol PX.

Bound P-selectin is eluted from the S12 Affigel with 100 mL of 80%ethylene glycol, 1 mM MES pH 6.0, 0.01% Lubrol PX. Peak fractions withabsorbance at 280 nm are pooled. Eluates are dialyzed against TBS with0.05% Lubrol, then applied to a Mono Q column (FPLC from Pharmacia). Theconcentrated protein is step eluted with 2 M NaCl, 20 mM Tris pH 7.5(plus 0.05% Lubrol PX for the membrane fraction). Peak fractions aredialyzed into TBS pH 7.5 (plus 0.05% Lubrol PX for the membranefraction).

P-selectin is plated at 5 micrograms/mL and the control proteins: humanserum albumin (Alb), platelet glycoprotein IIb/IIIa (IIb), vonWillebrand factor (vWF), fibrinogen (FIB), thrombomodulin (TM), gelatin(GEL) or human serum (HS), are added at 50 micrograms/mL. All wells areblocked for 2 hours at 22° C. with 300 microliters HBSS containing 10mg/mL HSA, then washed three times with HBSS containing 0.1% Tween-20and once with HBSS. Cells (2×10⁵ per well) are added to the wells andincubated at 22° C. for 20 minutes. The wells are then filled withHBSS/HSA, sealed with acetate tape (Dynatech), and centrifuged invertedat 150 g for 5 minutes. After discarding nonadherent cells andsupernates, the contents of each well are solubilized with 200microliters 0.5% hexadecyltrimethylammonium bromide, Sigma, in 50 mMpotassium phosphate, pH. 6.0, and assayed for myeloperoxidase activity,Ley, et al., Blood 73, 1324-1330 (1989). The number of cells bound isderived from a standard curve of myeloperoxidase activity versus numbersof cells. Under all assay conditions, the cells release less than 5% oftotal myeloperoxidase and lactate dehydrogenase. Inhibition is read as alower percent adhesion, so that a value of 5% means that 95% of thespecific adhesion was inhibited.

Clinical Applications

Since the selectins have several functions related to leukocyteadherence, inflammation, and coagulation, compounds which interfere withbinding of P-selectin, E-selectin or L-selectin can be used to modulatethese responses.

For example, the peptides can be used to competitively inhibit leukocyteadherence by competitively binding to P-selectin receptors on thesurface of leukocytes. This kind of therapy would be particularly usefulin acute situations where effective, but transient, inhibition ofleukocyte-mediated inflammation is desirable. Chronic therapy byinfusion of the peptides may also be feasible in some circumstances.

An inflammatory response may cause damage to the host if unchecked,because leukocytes release many toxic molecules that can damage normaltissues. These molecules include proteolytic enzymes and free radicals.Examples of pathological situations in which leukocytes can cause tissuedamage include injury from ischemia and reperfusion, bacterial sepsisand disseminated intravascular coagulation, adult respiratory distresssyndrome, tumor metastasis, rheumatoid arthritis and atherosclerosis.

Reperfusion injury is a major problem in clinical cardiology.Therapeutic agents that reduce leukocyte adherence in ischemicmyocardium can significantly enhance the therapeutic efficacy ofthrombolytic agents. Thrombolytic therapy with agents such as tissueplasminogen activator or streptokinase can relieve coronary arteryobstruction in many patients with severe myocardial ischemia prior toirreversible myocardial cell death. However, many such patients stillsuffer myocardial neurosis despite restoration of blood flow. This"reperfusion injury" is known to be associated with adherence ofleukocytes to vascular endothelium in the ischemic zone, presumably inpart because of activation of platelets and endothelium by thrombin andcytokines that makes them adhesive for leukocytes (Romson et al.,Circulation 67: 1016-1023 (1983)). These adherent leukocytes can migratethrough the endothelium and destroy ischemic myocardium just as it isbeing rescued by restoration of blood flow.

There are a number of other common clinical disorders in which ischemiaand reperfusion results in organ injury mediated by adherence ofleukocytes to vascular surfaces, including strokes; mesenteric andperipheral vascular disease; organ transplantation; and circulatoryshock (in this case many organs might be damaged following restorationof blood flow).

Bacterial sepsis and disseminated intravascular coagulation often existconcurrently in critically ill patients. They are associated withgeneration of thrombin, cytokines, and other inflammatory mediators,activation of platelets and endothelium, and adherence of leukocytes andaggregation of platelets throughout the vascular system.Leukocyte-dependent organ damage is an important feature of theseconditions.

Adult respiratory distress syndrome is a devastating pulmonary disorderoccurring in patients with sepsis or following trauma, which isassociated with widespread adherence and aggregation of leukocytes inthe pulmonary circulation. This leads to extravasation of large amountsof plasma into the lungs and destruction of lung tissue, both mediatedin large part by leukocyte products.

Two related pulmonary disorders that are often fatal are inimmunosuppressed patients undergoing allogeneic bone marrowtransplantation and in cancer patients suffering from complications thatarise from generalized vascular leakage resulting from treatment withinterleukin-2 treated LAK cells (lymphokine-activated lymphocytes). LAKcells are known to adhere to vascular walls and release products thatare presumably toxic to endothelium. Although the mechanism by which LAKcells adhere to endothelium is now known, such cells could potentiallyrelease molecules that activate endothelium and then bind to endotheliumby mechanisms similar to those operative in neutrophils.

Tumor cells from many malignancies (including carcinomas, lymphomas, andsarcomas) can metastasize to distant sites through the vasculature. Themechanisms for adhesion of tumor cells to endothelium and theirsubsequent migration are not well understood, but may be similar tothose of leukocytes in at least some cases. The association of plateletswith metastasizing tumor cells has been well described, suggesting arole for platelets in the spread of some cancers. Recently, it wasreported that P-selectin binds to tumor cells in a variety of humancarcinoma tissue sections (colon, lung, and breast), and that P-selectinbinds to the cell surface of a number of cell lines derived from variouscarcinomas, but not from melanomas. Aruggo, A., et al., Proc. Natl.Acad. Sci. USA, 89, 2292-2296 (1992). Aruggo et al. also referenceearlier work suggesting that E-selectin might be involved in tumormetastasis by mediating the adhesion of a colon carcinoma cell line(HT-20) to activated endothelial cells in vitro. Platelet-leukocyteinteractions are believed to be important in atherosclerosis. Plateletsmight have a role in recruitment of monocytes into atheroscleroticplaques; the accumulation of monocytes is known to be one of theearliest detectable events during atherogenesis. Rupture of a fullydeveloped plaque may not only lead to platelet deposition and activationand the promotion of thrombus formation, but also the early recruitmentof neutrophils to an area of ischemia.

Another area of potential application is in the treatment of rheumatoidarthritis.

The criteria for assessing response to therapeutic modalities employingthese peptides, and, hence, effective dosages of the peptides of thisinvention for treatment, are dictated by the specific condition and willgenerally follow standard medical practices. For example, the criteriafor the effective dosage to prevent extension of myocardial infarctionwould be determined by one skilled in the art by looking at markerenzymes of myocardial necrosis in the plasma, by monitoring theelectrocardiogram, vital signs, and clinical response. For treatment ofacute respiratory distress syndrome, one would examine improvements inarterial oxygen, resolution of pulmonary infiltrates, and clinicalimprovement as measured by lessened dyspnea and tachypnea. For treatmentof patients in shock (low blood pressure), the effective dosage would bebased on the clinical response and specific measurements of function ofvital organs such as the liver and kidney following restoration of bloodpressure. Neurologic function would be monitored in patients withstroke. Specific tests are used to monitor the functioning oftransplanted organs; for example, serum creatinine, urine flow, andserum electrolytes in patients undergoing kidney transplantation.

Diagnostic Reagents

The peptides can also be used for the detection of human disorders inwhich the ligands for the selectins might be defective. Such disorderswould most likely be seen in patients with increased susceptibility toinfections in which leukocytes might not be able to bind to activatedplatelets or endothelium. Cells to be tested, usually leukocytes, arecollected by standard medically approved techniques and screened.Detection systems include ELISA procedures, binding of radiolabeledantibody to immobilized activated cells, flow cytometry, or othermethods known to those skilled in the art. Inhibition of binding in thepresence and absence of the lectin domain peptides can be used to detectdefects or alterations in selectin binding. For selectins, suchdisorders would most likely be seen in patients with increasedsusceptibility to infections in which leukocytes would have defectivebinding to platelets and endothelium because of deficient leukocyteligands for P-selectin.

The peptide is labeled radioactively, with a fluorescent tag,enzymatically, or with electron dense material such as gold for electronmicroscopy. The cells to be examined, usually leukocytes, are incubatedwith the labeled peptides and binding assessed by methods describedabove with antibodies to P-selectin, or by other methods known to thoseskilled in the art. If ligands for P-selectin are also found in theplasma, they can also be measured with standard ELISA orradioimmunoassay procedures, using labeled P-selectin-derived peptideinstead of antibody as the detecting reagent.

The peptides can also be useful in in vivo imaging of concentrations ofcells bearing selectin ligands. Cells expressing selectin ligands whoseabnormally high local concentrations or presence within the body such ascancer cells, is indicative of a disorder can be imaged using labeledpeptides. These labels may be either intrinsic or extrinsic to thestructure of the specific selectin peptide and may include, but not belimited to high energy emitters such as ¹¹¹ In or non-radioactive denseatoms to enhance x-ray contrast.

The following examples are presented to illustrate, not limit, theinvention. In the examples and throughout the specification, parts areby weight unless otherwise indicated.

EXAMPLE I:Glutamyl-tyrosyl-leucyl-asparaginyl-serinyl-isoleucyl-leucyl-serinyl-tyrosyl-serinyl-prolinyl-serinyl-tyrosyl-tyrosyl-tryptophan-amide(SEQ ID NO:23)

The peptide was prepared on an ABI Model 431A Peptide Synthesizer usingVersion 1.12 of the standard Boc software. 4-methyl benzhydrylamineresin (625 mg) was used in the synthesis. The final weight of the resinwas 2.2 g.

The peptide was cleaved from the resin (2.2 g) using 22 mL of HF and 2.2mL of anisole for 60 min at 0° C. The resin was washed with ether andthe peptide extracted with 50% TFA/CH₂ Cl₂ to give 1.65 g of crudepeptide. The peptide was deformylated with 50 mL of 2% aqueouspiperidine at 4° C. for 2 hours.

The crude peptide (1.60 g) was purified on a Vydac C-18 column (15μ,5×25 cm) eluting with a 20-60% gradient of 80% acetonitrile in 0.1% TFAover 120 min at a flow rate of 15 mL per min. Fractions were collected,analyzed by HPLC and pure fractions pooled and lyophilized to give 49mg. Amino acid analysis: Asx 1.01 (1), Glx 0.97 (1), Ile 0.98 (1), Leu2.07 (2), Pro 1.01 (1), Ser 2.69 (4), Tyr 3.30 (4). FAB/MS: MH⁺ 1884.9

EXAMPLE II:Aspartyl-tyrosyl-leucyl-asparginyl-lysyl-valyl-leucyl-prolyl-tyrosyl-tyrosyl-serinyl-serinyl-tyrosyl-tyrosyl-valine-amide(SEQ ID NO:1)

The peptide was prepared on an ABI Model 431A Peptide Synthesizer usingVersion 1.12 of the standard Boc software. 4-methyl benzhydrylamineresin (0.631 g, 0.5 mmol) was used in the synthesis. The final weight ofthe resin was 2.476 g.

The peptide was cleaved from the resin (2.389 g) using 24 mL of HF and2.4 mL of anisole for 60 min at 0° C. The resin was washed with etherand the peptide extracted with a 1:1 solution of TFA/CH₂ Cl₂ to give1.044 g of crude peptide.

The crude peptide (944 mg) was purified on a Vydac C-18 column (15μ,5.0×25 cm) by using four 230 mg injections, eluting with a 15-75%gradient of 80% acetonitrile in 0.1% TFA over 120 min at a flow rate of15 per min. Fractions were collected, analyzed by HPLC and purefractions pooled and lyophilized to give 238 mg. Amino acid analysis:Asx 2.03 (2), Leu 2.04 (2), Lys 0.97 (1), Pro 1.01 (1), Ser 1.46 (2),Tyr 4.64 (5), Val 1.97 (2). FAB/MS: MH⁺ 1886

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 42    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 15 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - Asp Tyr Leu Asn Lys Val Leu Pro Tyr Tyr Se - #r Ser Tyr Tyr Val    #                 15    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 14 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Tyr Leu Asn Lys Val Leu Pro Tyr Tyr Ser Se - #r Tyr Tyr Val    #                 10    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 13 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - Leu Asn Lys Val Leu Pro Tyr Tyr Ser Ser Ty - #r Tyr Val    #                 10    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 12 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - Asn Lys Val Leu Pro Tyr Tyr Ser Ser Tyr Ty - #r Val    #                 10    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 11 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - Lys Val Leu Pro Tyr Tyr Ser Ser Tyr Tyr Va - #l    #                 10    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 14 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - Asp Tyr Leu Asn Lys Val Leu Pro Tyr Tyr Se - #r Ser Tyr Tyr    #             14  10    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 9 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    - Val Leu Pro Tyr Tyr Ser Ser Tyr Tyr      1               5    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 8 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    - Leu Pro Tyr Tyr Ser Ser Tyr Tyr      1               5    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 9 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    - Lys Thr Leu Pro Phe Ser Ser Tyr Tyr      1               5    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    - Val Leu Pro Tyr Tyr Ser Ser Tyr Tyr Val    #                 10    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 8 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    - Val Leu Pro Tyr Tyr Ser Ser Tyr      1               5    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    - Lys Val Leu Ala Tyr Tyr Ser Ser Tyr Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    - Lys Val Leu Pro Ala Tyr Ser Ser Tyr Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    - Lys Val Leu Pro Tyr Tyr Ser Ala Tyr Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    - Lys Val Leu Pro Tyr Tyr Ser Ser Ala Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    - Lys Val Leu Pro Tyr Tyr Ser Thr Tyr Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    - Lys Val Leu Pro Tyr Tyr Thr Ser Tyr Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    - Asp Val Leu Pro Tyr Tyr Ser Ser Tyr Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    - Lys Val Leu Pro Tyr Gly Ser Ser Tyr Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    - Lys Val Leu Pro Arg Tyr Ser Ser Tyr Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    - Asn Thr Leu Pro Tyr Ser Pro Tyr Tyr Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    - Lys Val Gln Pro Tyr Tyr Ser Ser Tyr Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 15 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    - Glu Tyr Leu Asn Ser Ile Leu Ser Tyr Ser Pr - #o Ser Tyr Tyr Trp    #                 15    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 15 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 3    #cysteine forms disulfide bondN:                   with cyst - #eine at position 13    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    - Asp Tyr Cys Asn Lys Val Leu Pro Tyr Tyr Se - #r Ser Cys Tyr Val    #                 15    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 14 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 2    #cysteine forms disulfide bondN:                   with cyst - #eine at position 12    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    - Tyr Cys Asn Lys Val Leu Pro Tyr Tyr Ser Se - #r Cys Tyr Val    #                 10    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 14 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 3    #cysteine forms disulfide bondN:                   with cyst - #eine at position 13    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    - Asp Tyr Cys Asn Lys Val Leu Pro Tyr Tyr Se - #r Ser Cys Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 13 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 3    #cysteine forms disulfide bondN:                   with cyst - #eine at position 13    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    - Asp Tyr Cys Asn Lys Val Leu Pro Tyr Tyr Se - #r Ser Cys    #                 10    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 13 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 2    #cysteine forms disulfide bondN:                   with cyst - #eine at position 12    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    - Tyr Cys Asn Lys Val Leu Pro Tyr Tyr Ser Se - #r Cys Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:29:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 13 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 1    #cysteine forms disulfide bondN:                   with cyst - #eine at position 11    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    - Cys Asn Lys Val Leu Pro Tyr Tyr Ser Ser Cy - #s Tyr Val    #                 10    - (2) INFORMATION FOR SEQ ID NO:30:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 12 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 1    #cysteine forms disulfide bondN:                   with cyst - #eine at position 11    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    - Cys Asn Lys Val Leu Pro Tyr Tyr Ser Ser Cy - #s Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:31:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 11 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 1    #cysteine forms disulfide bondN:                   with cyst - #eine at position 11    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    - Cys Asn Lys Val Leu Pro Tyr Tyr Ser Ser Cy - #s    #                 10    - (2) INFORMATION FOR SEQ ID NO:32:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 15 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 7    #cysteine forms disulfide bondN:                   with cyst - #eine at position 12    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    - Asp Tyr Leu Asn Lys Val Cys Pro Tyr Tyr Se - #r Cys Tyr Tyr Val    #                 15    - (2) INFORMATION FOR SEQ ID NO:33:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 14 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 6    #cysteine forms disulfide bondN:                   with cyst - #eine at position 11    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    - Tyr Leu Asn Lys Val Cys Pro Tyr Tyr Ser Cy - #s Tyr Tyr Val    #                 10    - (2) INFORMATION FOR SEQ ID NO:34:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 13 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 5    #cysteine forms disulfide bondN:                   with cyst - #eine at position 10    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    - Leu Asn Lys Val Cys Pro Tyr Tyr Ser Cys Ty - #r Tyr Val    #                 10    - (2) INFORMATION FOR SEQ ID NO:35:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 12 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 4    #cysteine forms disulfide bondN:                   with cyst - #eine at position 9    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    - Asn Lys Val Cys Pro Tyr Tyr Ser Cys Tyr Ty - #r Val    #                 10    - (2) INFORMATION FOR SEQ ID NO:36:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 11 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 3    #cysteine forms disulfide bondN:                   with cyst - #eine at position 8    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    - Lys Val Cys Pro Tyr Tyr Ser Cys Tyr Tyr Va - #l    #                 10    - (2) INFORMATION FOR SEQ ID NO:37:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 14 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 7    #cysteine forms disulfide bondN:                   with cyst - #eine at position 12    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    - Asp Tyr Leu Asn Lys Val Cys Pro Tyr Tyr Se - #r Cys Tyr Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:38:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 13 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 7    #cysteine forms disulfide bondN:                   with cyst - #eine at position 12    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    - Asp Tyr Leu Asn Lys Val Cys Pro Tyr Tyr Se - #r Cys Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:39:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 12 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 7    #cysteine forms disulfide bondN:                   with cyst - #eine at position 12    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    - Asp Tyr Leu Asn Lys Val Cys Pro Tyr Tyr Se - #r Cys    #                 10    - (2) INFORMATION FOR SEQ ID NO:40:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 13 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 6    #cysteine forms disulfide bondN:                   with cyst - #eine at position 11    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    - Tyr Leu Asn Lys Val Cys Pro Tyr Tyr Ser Cy - #s Tyr Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:41:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 12 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 5    #cysteine forms disulfide bondN:                   with cyst - #eine at position 10    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    - Leu Asn Lys Val Cys Pro Tyr Tyr Ser Cys Ty - #r Tyr    #                 10    - (2) INFORMATION FOR SEQ ID NO:42:    -      (i) SEQUENCE CHARACTERISTICS:    #acid residuesLENGTH: 11 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE:    #terminatedA) DESCRIPTION: Amide    -     (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 5    #cysteine forms disulfide bondN:                   with cyst - #eine at position 10    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    - Leu Asn Lys Val Cys Pro Tyr Tyr Ser Cys Ty - #r    #                 10    __________________________________________________________________________

What is claimed is:
 1. A peptide having a formula of:

    R.sup.1 --X.sup.1 --A'--B'--C'--D'--E'--F'--G'--Y.sup.1 --R.sup.2

or a pharmaceutically acceptable acid- or base-addition salt thereof,wherein: A' is D- or L-leucine, D- or L-threonine, or D- or L-glutamine;B' is D- or L-proline, D- or L-serine, D- or L-alanine, D- or L-valine,D- or L-isoleucine, D- or L-leucine, 2-aminobutyric acid, or null(signifying no amino acid); C' is D- or L-tyrosine, D- or L-proline, D-or L-arginine, or D- or L-alanine; D' is D- or L-tyrosine, D- orL-phenylalanine, D- or L-serine or glycine; E' is D- or L-serine, D- orL-threonine, or D- or L-proline; F' is D- or L-serine, D- or L-tyrosine,D- or L-alanine, or D- or L-threonine; G' is D- or L-tyrosine, or D- orL-alanine; X¹ is independently, a linear chain of from 0 to 6 aminoacids; Y¹ is independently, a linear chain of from 0 to 3 amino acids;R¹ is H (signifying a free-terminal amino group), formyl, lower alkyl,aryl, lower alkanoyl, aroyl, alkyloxycarbonyl, aryloxycarbonyl ordesamino (signifying no alpha amino group on the N-terminal amino acid);and R² is H (signifying descarboxy where the α carboxyl group in theC-terminal amino acid is absent), OR³, or NR⁴ R⁵ ; R³ is H (signifying afree carboxylic acid on a C-terminal carboxyl group) or lower alkyl oraryl; and R⁴ and R⁵ are each selected independently from H, lower alkyl,and aryl, or taken together are a methylene chain of 4-8 methylenegroups (--(CH₂)_(n) -- where n=4 to 8); provided that, when X¹--A'--B'--C'--D'--E'--F'--G'--Y¹ is DYLNKVLPYYSSYYW, then R² is NH₂. 2.A peptide of claim 1 wherein X¹ is selected from a group consistingof:Glu-Tyr-Leu-Asn-Ser-Ile, Asp-Tyr-Leu-Asn-Lys-X⁴, where X⁴ is selectedfrom the group consisting of Val, Pro, Gly, Ser and Asp;Tyr-Leu-Asn-Lys-Val, Leu-Asn-Lys-Val, Asn-Lys-Val, Asp-Val, Lys-Val,Asn-Thr, Val, Lys or null (signifying no amino acid),wherein each of theabove-designated amino acids may be a D- or L- amino acid.
 3. A peptideof claim 1 wherein Y¹ is selected from a group consisting of Tyr,Tyr-Val, Tyr-Trp, X⁵ -X⁶ -Val-, where X⁵ and X⁶ are each independentlyan aromatic or hydrophobic amino acid; and null (signifying no aminoacid), where each of the amino acids may be a D- or L- amino acid.
 4. Apeptide of claim 1 where R¹ is H and R² is NH₂.
 5. A peptide of claim 1wherein:A' is D- or L-leucine, D- or L-threonine, or D- or L-glutamine;B' is D- or L-proline, D- or L-serine, or D- or L-alanine; C' is D- orL-tyrosine, D- or L-proline, D- or L-arginine, D- or L-alanine, or null(signifying no amino acid); D' is D- or L-tyrosine, D- orL-phenylalanine, D- or L-serine or glycine; E' is D- or L-serine, D- orL-threonine, or D- or L-proline; F' is D- or L-serine, D- or L-tyrosine,D- or L-alanine, or D- or L-threonine; G' is D- or L-tyrosine, or D- orL-alanine; X¹ is a linear chain of from 0 to 6 amino acids; Y¹ is alinear chain of from 0 to 2 amino acids; R¹ is H (signifying afree-terminal amino group), formyl, lower alkyl, aryl, lower alkanoyl,aroyl, alkyloxycarbonyl, aryloxycarbonyl or desamino (signifying noalpha amino group on the N-terminal amino acid); and R² is H (signifyingdescarboxy where the α carboxyl group in the C-terminal amino acid isabsent) , OR³, or NR⁴ R⁵ ; R³ is H (signifying a free carboxylic acid ona C-terminal carboxyl group), lower alkyl or aryl; R⁴ and R⁵ are eachselected independently from H, lower alkyl, and aryl, or taken togetherare a methylene chain of 4-8 methylene groups (--(CH₂)_(n) -- where n=4to 8), provided that, when X¹ --A'--B'--C'--D'--E'--F'--G'--Y¹ isDYLNKVLPYYSSYYW, then R² is NH₂.
 6. A peptide of claim 5 wherein X¹ isselected from the group consisting of:Glu-Tyr-Leu-Asn-Ser-IleAsp-Tyr-Leu-Asn-Lys-Val, Tyr-Leu-Asn-Lys-Val, Leu-Asn-Lys-Val,Asn-Lys-Val, Asn-Thr, Lys-Val, Asp-Val Val, Lys or null (signifying noamino acid).
 7. A peptide of claim 5 wherein Y¹ is selected from a groupconsisting of Tyr, Tyr-Val, Tyr-Trp, or null (signifying no amino acid).8. A peptide of claim 6 wherein Y¹ is selected from a group consistingof Tyr, Tyr-Val, Tyr-Trp, or null (signifying no amino acid).
 9. Apeptide of claim 5 whereinA' is D- or L- leucine or D- or L-glutamine;B' is D- or L- proline or D- or L-serine; C' is D- or L-tyrosine, D- orL-alanine, D- or L-arginine, or null (signifying no amino acid); D' isD- or L-tyrosine, D- or L-phenylalanine, D- or L-serine or glycine; E'is D- or L-serine, D- or L-threonine or D- or L-proline; F' is D- orL-serine, D- or L-threonine, D- or L-alanine or D- or L-tyrosine; G' isD- or L-tyrosine or D- or L-alanine; X¹ is a linear amino acid chainselected from the group consisting of Glu-Tyr-Leu-Asn-Ser-Ile,Asp-Tyr-Leu-Asn-Lys-Val, Tyr-Leu-Asn-Lys-Val, Leu-Asn-Lys-Val,Asn-Lys-Val, Asn-Thr, Lys-Val, Asp-Val, Lys-Thr, Val, and null(signifying no amino acid); Y¹ is a linear amino acid chain selectedfrom the group consisting of Tyr, Val, Tyr-Val, Tyr-Trp, and null(signifying no amino acid); R¹ is H (signifying a free-terminal aminogroup), formyl, lower alkyl, aryl, lower alkanoyl, aroyl,alkyloxycarbonyl, aryloxycarbonyl or desamino (signifying no alpha aminogroup on the N-terminal amino acid); R² is H (signifying descarboxywhere the α-carboxyl group in the C-terminal amino acid is absent), OR³,or NR⁴ R⁵ ; R³ is H (signifying a free carboxylic acid on a C-terminalcarboxyl group), lower alkyl or aryl; R⁴ and R⁵ are each selectedindependently from H, lower alkyl, and aryl, or taken together are amethylene chain of 4-8 methylene groups (--(CH₂)_(n) -- where n=4 to 8);provided that, when X¹ --A'--B'--C'--D'--E'--F'--G'--Y¹ isDYLNKVLPYYSSYYW, then R² is NH₂.
 10. A peptide of claim 5 where R¹ is Hand R² is NH₂.
 11. A peptide of claim 9 where R¹ is H and R² is NH₂. 12.A peptide of claim 1 selected from the group consisting of:

    __________________________________________________________________________    (SEQ ID NO: 1)            Asp--Tyr--Leu--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--T            yr--Val-NH.sub.2 ;    (SEQ ID NO: 2)            Tyr--Leu--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr--V            al-NH.sub.2 ;    (SEQ ID NO: 3)            Leu--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr--Val-NH            .sub.2 ;    (SEQ ID NO: 4)            Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr--Val-NH.sub.            2 ;    (SEQ ID NO: 5)            Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr--Val-NH.sub.2 ;    (SEQ ID NO: 6)            Asp--Tyr--Leu--Asn--Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--T            yr-NH.sub.2 ;    (SEQ ID NO: 7)            Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 8)            Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 9)            Lys--Thr--Leu--Pro--Phe--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 10)            Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr--Val-NH.sub.2 ;    (SEQ ID NO: 11)            Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr-NH.sub.2 ;    (SEQ ID NO: 12)            Lys--Val--Leu--Ala--Tyr--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 13)            Lys--Val--Leu--Pro--Ala--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 14)            Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ala--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 15)            Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Ala--Tyr-NH.sub.2 ;    (SEQ ID NO: 16)            Lys--Val--Leu--Pro--Tyr--Tyr--Ser--Thr--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 17)            Lys--Val--Leu--Pro--Tyr--Tyr--Thr--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 18)            Asp--Val--Leu--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 19)            Lys--Val--Leu--Pro--Tyr--Gly--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 20)            Lys--Val--Leu--Pro--Arg--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 21)            Asn--Thr--Leu--Pro--Tyr--Ser--Pro--Tyr--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 22)            Lys--Val--Gln--Pro--Tyr--Tyr--Ser--Ser--Tyr--Tyr-NH.sub.2 ;    (SEQ ID NO: 23)            Glu--Tyr--Leu--Asn--Ser--Ile--Leu--Ser--Tyr--Ser--Pro--Ser--Tyr--T            yr--Trp-NH.sub.2.    __________________________________________________________________________


13. A pharmaceutical composition comprising at least one peptide ofclaim 1 in an amount effective to inhibit cellular adherence and apharmaceutically acceptable carrier or diluent.
 14. The pharmaceuticalcomposition of claim 13 wherein said pharmaceutically acceptable carrieror diluent is acceptable for parenteral administration.
 5. Thepharmaceutical composition of claim 13 wherein said pharmaceuticallyacceptable carrier or diluent is acceptable for oral administration. 16.A peptide of claim 1 wherein each of A', B', C', D', E', F', and G' isan L-amino acid, and wherein X¹ and Y¹ comprise L-amino acids.
 17. Apeptide of claim 2 wherein each of A', B', C', D', E', F', and G' is anL-amino acid, and wherein X¹ and Y¹ comprise L-amino acids.
 18. Apeptide of claim 3 wherein each of A', B', C', D', E', F', and G' is anL-amino acid, and wherein X¹ and Y¹ comprise L-amino acids.
 19. Apeptide of claim 9 wherein each of A', B', C', D', E', F', and G' is anL-amino acid, and wherein X¹ and Y¹ comprise L-amino acids.
 20. Apeptide of claim 12 wherein each of A', B', C', D', E', F', and G' is anL-amino acid, and wherein X¹ and Y¹ comprise L-amino acids.
 21. Apeptide of claim 13 wherein each of A', B', C', D', E', F', and G' is anL-amino acid, and wherein X¹ and Y¹ comprise L-amino acids.
 22. Apeptide of claim 15 wherein each of A', B', C', D', E', F', and G' is anL-amino acid, and wherein X¹ and Y¹ comprise L-amino acids.
 23. Apharmaceutical composition comprising at least one peptide of claim 21in an amount effective to inhibit cellular adherence and apharmaceutically acceptable carrier or diluent.